Multifunctional Nanoparticles as Nanocarrier for Vincristine Sulfate

Jan 24, 2014 - Amir Roointan , Sedigheh Kianpour , Fatemeh Memari , Molood Gandomani , Seyed Mohammad Gheibi Hayat , Soliman Mohammadi-Samani...
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Article pubs.acs.org/molecularpharmaceutics

Multifunctional Nanoparticles as Nanocarrier for Vincristine Sulfate Delivery To Overcome Tumor Multidrug Resistance Yuan Wang,†,‡ Limei Dou,† Huijuan He,† Yi Zhang,† and Qi Shen*,† †

School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China College of Pharmaceutical Science, Zhejiang Chinese Medical University, 548 Bingwen Road, Hangzhou 310053, P. R. China

‡

ABSTRACT: Multifunctional nanoparticles, Fol/R7 NPs, based on pH-sensitive PLGA-PEG−folate and cell penetrating peptide R7-conjugated PLGA-PEG, were constructed for targeting vincristine sulfate (VCR) to tumor and overcoming multidrug resistance (MDR). In this study, the pH-triggered VCR release was 65.6% during 8 h in pH 5.0, but only 35.8% in pH 7.4, demonstrating that a large amount of VCR released rapidly at weak acidic environment. The VCR-Fol/R7 NPs could significantly enhance cellular uptake and cytotoxicity in MCF-7 and MCF-7/Adr cells when compared to the nanoparticles solely modified by folate or R7. With folate receptor-mediated endocytosis and strong intracellular penetration, VCR-Fol/R7 NPs increased drug accumulation in resistant tumor cells by escaping P-glycoprotein mediated drug efflux. In vivo imaging suggested the active targeting attributed to pH sensitivity and folate receptor-mediated effect could improve tumor targeting efficacy. Indeed, VCR-Fol/R7 NPs exhibited the stongest antitumor efficacy in vivo. Therefore, Fol/R7 NPs are an effective nanocarrier for delivering antitumor drug and overcoming multidrug resistance. KEYWORDS: nanoparticles, multifunctional, pH sensitive, vincristine sulfate, multidrug resistance

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INTRODUCTION Traditional chemotherapy is an indispensable approach for cancer treatment. However, its therapeutic efficacy is usually limited by two major challenges. (1) The occurrence of multidrug resistance (MDR) phenotypes leads to chemotherapeutic failure. P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells has been regarded as the major mechanism of MDR;1,2 it is capable of pumping a wide range of cancer chemotherapeutic agents outside the cells, leading to a low intracellular drug accumulation below the effective dose value.3−5 (2) Cancer chemotherapeutic agents can unselectively enter into both normal tissues and tumor tissues, resulting in undesirable side effects and even death of the patients.6 Vincristine sulfate (VCR), as an antimitotic chemotherapeutic agent, is widely used to treat different cancers, including malignant lymphoma, acute leukemia and breast cancer.7 However, its clinical application exhibits low absorption and fast elimination attributed to P-gp-mediated efflux, and causes side effects based on dose-limiting systemic toxicity. To overcome drug resistance, bypassing the P-gp-mediated drug efflux pumps through the effect of nanoparticle-mediated endocytosis is an effective strategy, besides inhibiting the P-gp expression in tumor cell membrane by some functional inhibitors. 8−10 To minimize side effects and improve therapeutic efficiency, multifunctional drug delivery systems are used to transport the drug to tumor precisely through passive and active targeting.6 In recent years, ligand-modified nanoparticles (NPs) as drug delivery systems have received © 2014 American Chemical Society

considerable attention. Folate receptor is overexpressed in a broad spectrum of human cancers, especially in gynecological cancers, such as ovarian, endometrial and breast cancers.11,12 Therefore, folate-modified polymeric carrier is utilized to achieve tumor-specific targeting and increase cellular uptake via receptor-mediated effect.13−15 Arginine-rich cell-penetrating peptides (CPPs), due to relatively simple structure, are widely used to improve cellular uptake efficiency,14,16,17 such as CPPmodified nanoparticles18 and anticancer drug−CPP conjugation.19 It has been shown that the pH values of the tumor extracellular (6.5) and intracellular (4.0−6.0) microenvironments are lower than that in normal tissue or blood (7.4).20,21 Thus, pH-sensitive drug-loading NPs can be designed to reduce drug leakage during transportation, and control drug release to specific position,22 enhancing the effect of active tumor targeting. Poly(lactic-co-glycolic acid) (PLGA) has excellent mechanical strength, biocompatibility and biodegradability, so it is widely used in nanodrug delivery systems to control drug release.23 However, both PLGA-based nanoparticles and ligandmodified nanoparticles are easily eliminated during circulation by the reticuloendothelial system (RES). To overcome these disadvantages, hydrophilic polymer polyethylene glycol (PEG) is used to modify the surface of nanoparticles.24 Received: Revised: Accepted: Published: 885

September 14, 2013 December 9, 2013 January 24, 2014 January 24, 2014 dx.doi.org/10.1021/mp400547u | Mol. Pharmaceutics 2014, 11, 885−894

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Scheme 1. Schematic Representation of VCR-Fol/R7 NPs for Overcoming Multidrug Resistance of MCF-7/Adr Cells

Scheme 2. The Synthetic Route of pH-Sensitive PLGA-PEG−Folate

For these reasons, we constructed a novel multifunctional nanoparticle drug delivery system containing two functional polymers: pH-sensitive PLGA-PEG−folate [folate was conjugated to PLGA-PEG, which was synthesized by using hydrazone bond (-CN-NH-) as an acid-cleavable linkage to

couple PLGA to PEG]; PLGA-PEG-R7 [PLGA-PEG was modified with cell-penetrating peptides (R7)]. To evaluate the multifunctional nanoparticles (Fol/R7 NPs) in vitro and in vivo, different VCR-loaded nanoparticles were prepared by a modified double-emulsion (w/o/w) solvent evaporation 886

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nitrogen atmosphere to prepare pht-PEG-pht, which was then mixed with NH2-NH2 in ethanol for 5 days to produce NH2PEG-NH2 (Mw 3382 Da). HO-PLGA-COO-ph-CHN-NHph-COOH was synthesized by coupling HO-PLGA-COO-phCHO with 4-hydrazinobenzoic acid, the reaction was catalyzed by p-toluenesulfonic acid. HO-PLGA-COO-ph-CHO was obtained from chemical conjugation between p-hydroxybenzaldehyde and the activated PLGA-COOH. HO-PLGA-COO-phCHN-NH-ph-COOH (1.5 g, 0.1 mmol), activated by DCC (51.5 mg, 0.25 mmol), NHS (28.75 mg, 0.25 mmol) and Et3N (2 mL, 14.2 mmol), was added slowly to a mixture of NH2PEG-NH2 (78 mg, 20 mmol) and pyridine in DMSO under gentle stirring, then reacted for 60 h. Folate (1.5 g, 3 mmol), activated by DCC (94 mg, 0.45 mmol), NHS (52 mg, 0.45 mmol) and Et3N (5 mL, 35.5 mmol), was mixed with the above reaction product, pyridine and DCC (10 mg, 0.05 mmol) at room temperature under magnetic stirring for 60 h to prepare pH-sensitive PLGA-PEG−folate, then filtered, dialyzed (MWCO, 10 kDa) and dried under vacuum. The PLGAPEG-R7 was synthesized as previously described.14 Preparation of VCR-Loaded NPs. Four types of VCRloaded NPs were prepared: (1) VCR-P NPs (PLGA-mPEG 35 mg), (2) VCR-R7 NPs (PLGA-mPEG 30 mg, PLGA-PEG-R7 5 mg), (3) VCR-Fol NPs (PLGA-mPEG 30 mg, PLGA-PEG− folate 5 mg) and (4) VCR-Fol/R7 NPs (PLGA-mPEG 25 mg, PLGA-PEG−folate 5 mg, PLGA-PEG-R7 5 mg). All nanoparticles were prepared by the double-emulsion method28,29 with some modifications. In brief, 2.5 mg of VCR and 0.2 mg of DS were dissolved in 500 μL of Tris-HCl buffer (pH 6.8), and then slowly added to 4 mL of methylene dichloride/acetone (v:v = 1:1) containing 35 mg of polymeric carriers. The mixture was cooled in an ice bath and emulsified by sonication for 2 min at 90 W using a Scientz-IID ultrasonic probe (Ningbo Scientz Biotechnology Corp, China). 50 mL of 1.0% (w/v) PVA solution whose pH value was adjusted to 8−9 by Tris-HCl buffer was added to the primary emulsion and sonicated for 5 min to form a double emulsion. Organic solvents were removed by evaporating under reduced pressure at room temperature. The nanoparticles were collected by centrifugation at 15,000 rpm for 30 min, and washed three times with distilled water. The different formulations of Rh-123-loaded NPs and RBloaded NPs were prepared with the same procedure as described above, except the Rh-123 or RB was dissolved in 500 μL of Tris-HCl buffer. Characterization of VCR-Loaded NPs. The average particle size and zeta potential of the different VCR-loaded NPs were measured by dynamic light scatting (DLS, Zetasizer 3000, Malvern Instruments Ltd., Malvern, U.K.). All measurements were performed in triplicate. The morphology of VCRFol/R7 NPs was observed by transmission electron microscopy (TEM, JEM-2010, JEOL, Tokyo, Japan) with phosphotungstic acid solution (1.5%, w/v) staining. The drug encapsulation efficiency and drug loading were analyzed by the high-performance liquid chromatography method (HPLC, LC-20AT, Shimadzu, Japan) with a reverse phase C18-column (200 × 4.6 mm, 5 μm, 1 mL/min, Dikma Technologies, China). The lyophilized VCR-loaded NPs were dissolved in dimethylformamide (DMF) to release VCR. VCR was quantified by UV detection at 297 nm, and the mobile phase consisted of a mixture of 0.02 M aqueous dipotassium hydrogen phosphate−methanol (4:7, v/v, pH 4.7). The drug encapsulation efficiency (%) and drug loading (%) were calculated by the following equations:

method. The different pH values of release medium were selected to explore the pH-triggered drug release characteristic of VCR-Fol/R7 NPs. Human breast carcinoma cells were selected for the experimental research due to the clinically applicable cancer of VCR and the overexpression of folate receptors on the cell surfaces.25,26 The in vitro cytotoxicity and cellular uptake in MCF-7 and MCF-7/Adr cells were performed to evaluate the cytotoxic effect of different nanoparticles, and further explore its possible mechanism of overcoming the multidrug resistance (MDR). The VCR-Fol/R7 NPs possess pH-sensitive, targeting and penetrating effect (Scheme 1). So far, little literature has explored the tumortargeting and antitumor efficiency of this multifunctional nanoparticle (VCR-Fol/R7 NPs) on nude mice bearing MCF7/Adr tumors. Therefore, in vivo imaging and antitumor experiments were performed.

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MATERIALS AND METHODS Materials. PEG (Mw 3350 Da) and folate were obtained from Sinopharm Chemical Reagent Shanghai Co., Ltd. (Shanghai, China). PLGA (50:50, M w 15 kDa) and PLGA15000-mPEG2000 were purchased from Jinan Daigang Biology Tech Co., Ltd. (Jinan, Shandong Province, China). Vincristine sulfate (VCR) was purchased from Shanghai Anticancer Phytochemistry Co., Ltd. (Shanghai, China). Dextran sulfate sodium (DS, Mw 5000 Da) was obtained from Shanghai Xibao Biotech Co., Ltd. (Shanghai, China). NHydroxysuccinimide (NHS), triethylamine (Et3N), dimethyl sulfoxide (DMSO) and dicyclohexylcarbodiimide (DCC) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Folate-free RPMI 1640 medium was supplied by Gibco (Carlsbad, CA, USA). Phosphate buffered solution (PBS), fetal bovine serum (FBS), 0.25% trypsin−0.02% EDTA tetrasodium and penicillin−streptomycin were purchased from Biosun Biotechnology Co., Ltd. (Shanghai, China). 3(4,5-Dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine-123 (Rh-123) and rhodamine B (RB) were supplied by Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade. Cell Lines and Animals. MCF-7 cells (human breast carcinoma cell line) and MCF-7/Adr cells (multidrug resistant variant) were purchased from Shanghai Institute for Biology and Science (Shanghai, China). Cells were maintained in folatefree RPMI-1640 medium supplemented with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) and cultured in a 95% air humidified environment containing 5% CO2 at 37 °C. BALB/c nude mice (female, 5−6 weeks old, 20 ± 2 g) were supplied by Shanghai Laboratory Animal Center (Shanghai, China), and maintained in an animal laboratory under specific pathogen-free (SPF) conditions. All animal procedures were performed in compliance with the protocol evaluated and approved by the ethics committee of Shanghai Jiao Tong University. Synthesis of pH-Sensitive PLGA-PEG−Folate and PLGA-PEG-R7. The synthetic route of pH-sensitive PLGAPEG−folate is shown in Scheme 2. In brief, the polymer NH2PEG-NH2 was synthesized as previously described27 with modifications. SOCl2 (2.18 mL, 30 mmol) was added dropwise to a solution of PEG (20 g, 6 mmol) in 20 mL of methylbenzene, after 4 h of reaction at room temperature. The Cl-PEG-Cl obtained was further reacted with phthalimide potassium salt in DMF (100 mL) for 48 h at 120 °C under a 887

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Figure 1. Characterization of pH-sensitive PLGA-PEG−folate: FT-IR (A) and 1H NMR (B) spectra of PLGA-PEG−folate.

volume of fresh release medium. The specific concentration of released VCR was measured by HPLC. In Vitro Cytotoxicity Assays. To test the cytotoxicity of various VCR-loaded NPs, MCF-7 and MCF-7/Adr cells were seeded in a 96-well plate (5 × 103 cells/well) for 24 h, respectively. The seeding medium was then replaced with 100 μL of fresh medium containing blank NPs, F-VCR, VCR-P NPs, VCR-R7 NPs, VCR-Fol NPs and VCR-Fol/R7 NPs at various concentrations. After 48 h incubation, the cells were incubated for another 4 h with 20 μL of MTT solution (5 mg/ mL). After removing the culture medium, the resulting formazan crystal was dissolved in 100 μL of DMSO. The plates were measured at 490 nm using the microplate reader. All assays were performed with six parallel samples. Cellular Uptake. To study the cellular uptake and intracellular distribution of NPs, MCF-7 and MCF-7/Adr

encapsulation efficiency (%) = (Wentrapped /Wtotal) × 100% drug loading (%) = (Wentrapped /WNPs) × 100%

Wtotal is the total amount of VCR, Wentrapped is the amount of the encapsulated drug, and WNPs is the weight of the freezedried NPs. In Vitro Drug Release. The release of VCR from the VCRFol/R7 NPs was examined using the dialysis bag method. TrisHCl buffer solutions (pH 7.4) and acetic buffer solutions (ABS, pH 5.0) were used as the drug release media. Up to 2 mL of VCR-Fol/R7 NPs was added to dialysis bags (MWCO 3000 Da), which were sealed and then immersed in 30 mL of release medium with 100 rpm at 37 ± 0.5 °C. At predetermined time points, 300 μL of the solution outside the dialysis bag was withdrawn for analysis and then replaced with an equivalent 888

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cells were seeded on coverslips in 6-well culture plates (5 × 105 cells/well), respectively. After 24 h, the cells were treated with fresh medium containing Rh-123, Rh-123-P-NPs, Rh-123-R7 NPs, Rh-123-Fol NPs and Rh-123-Fol/R7 NPs at the same Rh123 concentration of 5 μg/mL, respectively, and cells were incubated for another 4 h. Afterward, the stained coverslips were washed three times with cold PBS and then observed under a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany). The cellular uptake of NPs was further confirmed by flow cytometer. The medium without Rh-123 was used as a blank control. Cells were washed with PBS, harvested in 0.5 mL of PBS and then measured by flow cytometer (FACScan, Becton Dickinson). In Vivo Imaging. Rhodamine B (RB) was encapsulated in nanoparticles as a fluorescent probe for investigating tumortargeting ability of the multifunctional nanoparticles. In order to prepare MCF-7/Adr tumor-bearing mice, MCF-7/Adr cells (5 × 106 cells) were injected subcutaneously in the right axilla of nude mice. Fifteen days after implantation, the tumorbearing mice were randomly assigned to three groups. RB-P NPs, RB-Fol NPs and RB-Fol/R7 NPs were respectively injected into mice through the tail vein at the same dose of RB (5 mg/kg). After treatment, the in vivo fluorescence imaging was carried out at predetermined time points, using eXplore Optix MX system (Advanced Research Technologies-GE Healthcare, Quebec, Canada) with excitation wavelength of 510 nm and emission wavelength of 600 nm. Mice were sacrificed at forty-eight hours postinjection, and tumors were collected and imaged. In Vivo Antitumor Effect. Tumor-bearing mice were established as described above. When the tumor volume grew to approximately 50 mm3, the mice were randomly assigned to the control (saline) group, F-VCR group, VCR-P NP group, VCR-Fol NP group and VCR-Fol/R7 NP group, each group containing five mice. The different formulations of VCR were given at the same dose of 1 mg/kg and administered intravenously through the tail vein every three days for seven times. The body weight and tumor size were measured and recorded every other day after administration. Tumor volume was calculated by the following equation:30 V = π/6 × longest diameter × (shortest diameter)2. Mice were sacrificed on the 21st day, and the weight of tumor was measured to evaluate antitumor efficacy. Statistical Analysis. All results were presented as mean ± standard deviation (SD) and were repeated at least three times. Student’s t test was performed to assessed the statistical significance of differences between two groups, and one-way ANOVA with Dunnett’s test was used for multiple comparisons; p < 0.05 was considered significant.

corresponded to multiple methylene groups in the PEG chain. The formation of an amide bond between the carboxyl group and the amino group was deduced from the presence of a slightly salient peak at 1683.80 cm−1. Folate was conjugated at the end of PLGA-PEG, which showed a prominent peak at 1763.68 and 1427.91 cm−1, corresponding to the -CO stretching in the polymers. The peaks at 1630.96 and 894.07 cm−1 could be attributed to hydrazone (-CHN-). To further detect the structure of the synthesized polymer, 1H NMR (in DMSO-d6, 300 M) was used. The spectrum is shown in Figure 1B. The peaks from 1.112 to 1.811 ppm were assigned to methyl hydrogen (-CH3) of PLGA segments; in addition, the peaks at 5.660 ppm, 5.294 ppm and 4.976 ppm belonged to the protons of hydroxyl (HO-CH2-CO-), methine (-CH) and methylene (-O-CH2-CO-) groups, respectively. The peaks at 3.484 ppm to 3.801 ppm belonged to the methene protons (-CH2-) repeatedly present in the PEG segments. The peaks at 7.522 ppm and 7.695 ppm belonged to the protons of hydrazone (-CHN-NH-), whereas the peaks located at 6.757 ppm belonged to the protons from the imino group (-NH-) of folate segments. The peaks at 4.594 ppm and 4.244 ppm corresponded to methylene hydrogen (-CH2-NH-) and methine hydrogen (-NH-CH-COOH). The peaks at 2.320 ppm and 1.891 to 2.097 ppm corresponded to the two different methylene hydrogen (-CH-CH2-CH2-COOH) groups of folate segments. These results confirmed the successful conjugation of the four units folate, PEG, PLGA and pH-sensitive hydrazone (-CHN-NH-) after several regular reactions, through which we obtained a polymer possessing both pH sensitivity and targeting function. Preparation and Characterization of VCR-Loaded NPs. Water−oil−water emulsion solvent evaporation method with modification was used. According to a previous report,31 DS, a negative polymer, was introduced to form an electrostatic complex with positive VCR. The optimized charge ratio was 1:1. PVA aqueous solution (pH 8−9) was used as external aqueous phase, and Tris-HCl buffer with pH 6.8 was selected as the solvent of the internal aqueous phase. Under optimal conditions, VCR-P NPs, VCR-R7 NPs, VCR-Fol NPs and VCR-Fol/R 7 NPs were successfully prepared. Table 1 Table 1. Physicochemical Characteristics of the Different VCR Nanoparticle Formulations (Mean ± SD, n = 3) group VCR-P NPs VCR-R7 NPs VCR-Fol NPs VCRFol/R7 NPs

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RESULTS AND DISCUSSION Synthesis and Characterization of pH-Sensitive PLGAPEG−Folate and PLGA-PEG-R7. The novel polymer, pHsensitive PLGA-PEG−folate, was designed using PEG as a hydrophilic block, PLGA as a hydrophobic block, folate as an active targeting group, and hydrazone (-CHN-NH-) as a pHsensitive group to perform a passive targeting function. The pH-sensitive PLGA-PEG−folate was characterized through FTIR (Figure 1A). In the infrared spectrum of the polymer pH-sensitive PLGA-PEG−folate, the bands 3333.13 cm−1 corresponded to imino (-NH-). A series of peaks between 2800 and 3050 cm−1 indicated that the C−H stretching

particle size (nm)

zeta potential (mV)

entrapment efficiency (%)

drug loading (%)

132.5 ± 6.2

−5.80 ± 1.43

52.8 ± 3.5

3.05 ± 0.19

138.2 ± 8.1

−7.26 ± 1.62

50.6 ± 3.9

2.98 ± 0.23

143.8 ± 9.3

−8.96 ± 1.83

49.4 ± 4.2

2.92 ± 0.25

149.3 ± 10.7

−9.12 ± 1.91

48.9 ± 4.4

2.87 ± 0.27

summarizes the average particle size, zeta potential, entrapment efficiency and drug loading of the various VCR-loaded NPs. The particle sizes of the modified VCR-loaded NPs slightly increased compared with that of unmodified VCR-P NPs, which is attributed to their modification with folate or R7. Furthermore, the molecular weights of PLGA and PEG were also important factors affecting the particle size. Increasing the molecular weight of PLGA increased viscosity of the polymer 889

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VCR-Fol/R7 NPs in the pH 7.4 solution was 35.8% in the first 8 h. In the following phases, a continued-release pattern was observed, but the release rate was slower, and no more than 62.7% of the total VCR was released after 60 h. However, the difference in the release behaviors of VCR-Fol/R7 NPs was noticeable in the pH 5.0 solution. The VCR showed a rapidrelease phase with a cumulative release of 65.6% in 8 h, reaching nearly 88.2% after 60 h. The result indicated that the release amount and rate of VCR from VCR-Fol/R7 NPs were improved by the acid-triggered breakage of hydrazone bonds in the acidic medium. The pH-sensitive hydrazone bonds (-C N-NH-) in the polymer were hydrolyzed easily, which facilitated the acidic medium to penetrate into the interior of the NPs, resulting in the burst release process. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of MCF-7 and MCF-7/Adr cells incubated with various VCRloaded NPs versus F-VCR at different drug concentrations was determined by the MTT method. Based on the result (Figure 4), the various VCR-loaded NPs showed a dose-dependent cytotoxicity against both MCF-7 and MCF-7/Adr cells. The resistant index of 62.07 for MCF-7/Adr cells was based on the cytotoxicity of F-VCR in MCF-7/Adr cells (IC50: 479.80 ± 8.62 μg/mL) compared to that in MCF-7 cells (IC50: 7.73 ± 0.72 μg/mL), indicating the drug resistant MCF-7/Adr cells expressed large amounts of P-gp compared with the drugsensitive MCF-7 cells. In view of P-gp-mediated efflux activity, a large dose of VCR was used to kill drug resistant cells. However, the IC50 of different VCR-loaded NPs was decreased from 479.80 μg/mL to 27.1 μg/mL in MCF-7/Adr cells; VCRFol/R7 NPs exhibited much higher cytoxicity (IC50: 27.1 ± 1.47 μg/mL) than that of F-VCR (IC50: 479.80 ± 8.62 μg/mL). The specific cytotoxicity of different VCR-loaded NPs followed the order VCR-Fol/R7 NPs > VCR-Fol NPs > VCR-R7 NPs > VCR-P NPs > F-VCR. This finding suggests that VCR-Fol/R7 NPs through the functional group remarkably enhanced the cellular uptake efficiency. When pH-sensitive nanoparticles enter into the acidic microenvironment of tumor cells, a large amount of drugs will be released rapidly,36−38 which timely offsets intracellular drug reduction caused by drug efflux transporter (P-gp), leading to reach the effective dose to kill drug-resistant cells.39,40 The cell viability of blank NPs was simultaneously examined, and the result indicated that the cell viability was 99.6% to 97.0%. This result suggested that the blank NPs did not cause evident cytotoxicity. Cellular Uptake. Since Rh-123 has been used as a specific substrate for efflux pump P-gp, 41 and showed green fluorescence, Rh-123 was selected to visualize cellular uptake and intracellular distribution of NPs instead of VCR. The

solutions, and increasing the PEG chain length extended molecular structure into the water phase, eventually leading to particle size increase.32−34 Therefore the relatively smaller molecular weights of PLGA15000 and PEG2000 were selected to synthesize functional polymers and further prepare multifunctional nanoparticles. Indeed, the particle sizes of different VCRloaded NPs were all below 200 nm. It is known that nanoparticles ranging from 100 to 200 nm are suitable for enhancing permeation and retention (EPR) effect within tumor vasculature and overcoming the fast elimination by kidneys.35 As shown in Figure 2, The TEM images of the VCR-Fol/R7 NPs demonstrated that particles with typical spherical shapes were successfully prepared by improved emulsion solvent evaporation method.

Figure 2. TEM photograph of VCR-Fol/R7 NPs.

In Vitro Drug Release. The release behaviors of VCR from the VCR-Fol/R7 NPs were observed in Tris-HCl buffer (pH 7.4). Owing to the pH-sensitive function of the Fol/R7 NPs, the other release medium (ABS, pH 5.0) was also selected to further evaluate the release profile of the VCR-Fol/R7 NPs. As shown in Figure 3, the cumulative release of VCR from the

Figure 3. In vitro release profile of VCR from VCR-Fol/R7 NPs in Tris-HCl buffer solution (pH 7.4) and acetic buffer solution (pH 5.0) at 37 ± 0.5 °C. Data are presented as mean ± SD (n = 3).

Figure 4. Cytotoxic activity of F-VCR, VCR-P NPs, VCR-R7 NPs, VCR-Fol NPs and VCR-Fol/R7 NPs to MCF-7 (A) and MCF-7/Adr cells (B) after incubation of 48 h. Data are presented as mean ± SD (n = 3). 890

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Figure 5. Confocal laser scanning microscopy (CLSM) (A) and flow cytometry analysis (B) of MCF-7 and MCF-7/Adr cells incubated with different Rh-123 formulations in the medium for 4 h, respectively.

MCF-7 cells displayed relatively stronger fluorescence intensity than MCF-7/Adr cells in the Rh-123 solution group (Figure 5A); the evidence also demonstrated the P-gp overexpressing in MCF-7/Adr cells. Compared to unmodified NPs, both Fol NPs and R7 NPs increased the uptake of Rh-123 in MCF-7 and MCF-7/Adr cells. The effect of the former was due to the folate-assisted endocytosis mechanism and acidic microenvironment-activated Rh-123 release;42 the effect of the latter lay in enhancing cell penetration by R7. As expected, Rh-123-Fol/R7 NPs exhibited the highest fluorescence intensity among different Rh-123 formulations in MCF-7 and MCF-7/Adr cells. Flow cytometry was employed to quantify the uptake of the NPs. As shown in Figure 5B, cells without Rh-123 were used as a control that showed the autofluorescence; different Rh-123 formulations were internalized by MCF-7 and MCF-7/Adr cells following the same order: Rh-123-Fol/R7 NPs > Rh-123-Fol NPs > Rh-123-R7 NPs > Rh-123-P NPs > Rh-123 solution. The specific geometric mean fluorescent intensities of Rh-123 solution group and Rh-123-Fol/R7 NP group were 272.3 and 680.1 in MCF-7 cells and 6.2 and 570.4 in MCF-7/Adr cells. The cellular uptake of Rh-123 solution in MCF-7 cells was 43.9-fold higher than that in MCF-7/Adr cells. The above results were consistent with CLSM data. Interestingly, the cellular uptake of Rh-123-Fol/R7 NPs in MCF-7 cells was only 1.2-fold that in MCF-7/Adr cells, demonstrating that Fol/R7 NPs can overcome drug resistance and enhance cellular uptake, further improving therapeutic efficacy in MDR tumor cells.42 In Vivo Imaging. Before testing the antitumor effect of multifunctional nanoparticles in vivo, the tumor-targeting efficiency of different nanoparticle formulations through in vivo imaging was evaluated. As shown in Figure 6, it was clear that for RB-Fol/R7 NP treated mice, the fluorescent signals at the tumor site were much stronger than those of the other two different nanoparticle formulations at all the time points after injection, demonstrating that the RB-Fol/R7 NPs could target to MCF-7/Adr tumor effectively and precisely. The specific

Figure 6. The in vivo imaging of mice bearing MCF-7/Adr tumors which were intravenously injected with RB-P NPs (a), RB-Fol NPs (b) and RB-Fol/R7 NPs (c) respectively at predetermined time points, with ex vivo imaging of the tumor at 48 h postinjection. The arrow indicates the MCF-7/Adr tumor.

tumor-targeting efficiency followed the order RB-Fol/R7 NPs > RB-Fol NPs > RB-P NPs. This result was also demonstrated by ex vivo imaging of tumors removed from tumor-bearing mice at 48 h postinjection (Figure 6). RB-P NPs were gradually increased in the tumor site within 48 h, which was connected with the passive targeting of unmodified NPs. The main mechanism was the permeation and retention (EPR) effect of NPs; it was a special phenomenon of tumor tissue associated with its pathophysiological and anatomical distinctions from normal tissue.35,43,44 Therefore, EPR effect was an important strategy for nanocarrier to improve the efficiency of drug delivery. Compared with RB-P NPs, relatively more RB-Fol NPs entered into tumor rapidly; this might be due to the combination of folate conjugation for 891

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Figure 7. In vivo antitumor effect of free VCR, VCR-P NPs, VCR-Fol NPs and VCR-Fol/R7 NPs on nude mice bearing MCF-7/Adr tumors (n = 5). (A) Picture of tumors of each group removed from the sacrificed mice at the end of the experiment. (B) Tumor volume changes after intravenous injection of various formulations of VCR (1 mg/kg). (C) Tumor weight at the end of the experiment. (D) Body weight changes of nude mice bearing MCF-7/Adr tumors after injection with various formulations of VCR. Each value represents the mean ± SD of five experiments (*, ** indicate P < 0.05, 0.01 versus the F-VCR group).

treated group (P < 0.05). The former result demonstrated that VCR-Fol NPs significantly improved the therapeutic efficiency in the presence of pH-sensitive PLGA-PEG−folate, because it enhanced the active accumulation, as well as increased the amount and rate of drug release in tumor tissues.48 The later result implied that PLGA-PEG-R7 played a critical role in penetrating into tumor cells and tissues and reducing escape from tumor into the circulatory system. These results are consistent with in vivo imaging data. The body weight was measured over the course of treatment to monitor the systemic toxicity. As shown in Figure 7D, the body weight of mice with F-VCR treatment showed an obvious decrease, and was reduced 6.22% of their original weight at the end of the experiment. It means that F-VCR exhibited toxicity in mice at a dose of 1 mg/kg. However, in the case of the VCRP NP, VCR-Fol NP, VCR-Fol/R7 NP and control (saline) groups, the body weight was increased by 6.86%, 8.82%, 17.96% and 26.44%, respectively, which suggested that they could reduce adverse side effects of VCR to some extent. The VCR-Fol/R7 NP group showed the lowest systemic toxicity in different VCR formulation treated groups. The above results were mainly because VCR-Fol/R7 NPs could reduce the distribution of VCR to normal tissues, and increase the distribution of VCR to tumor tissue simultaneously. In the control (saline) group, the body weight of nude mice gradually increased due to tumor weight and without adverse effects of VCR.22 Both the tumor inhibition rate and the result of systemic toxicity indicated that VCR-Fol/R7 NPs were effective and safe for treating the MCF-7/Adr tumor in nude mice.

receptor-mediated active targeting and acidic microenvironment-activated drug release,45,46 as well as EPR effect. When RB-Fol NPs distributed around the tumors, they could couple to folate receptor and then quickly internalize into tumor cells through endocytosis. The fluorescent signals of RB-Fol/R7 NP treated mice were clearly observed at tumor site in the first 4 h, and lasted strongest and longest. This was mainly because RBFol/R7 NPs targeted specifically to MCF-7/Adr tumor site via active targeting effect, and then entered into tumor tissues and cells through the cell-penetrating effect of R7.14,47 In Vivo Antitumor Effect. The antitumor efficacy of multifunctional nanoparticles was decided by measuring changes in tumor volume during the experiment and final tumor weight. The increased tumor size in different treatment groups over time can be seen in Figure 7B; based on the results, the tumor volume of the mice treated with different VCR formulations followed the order control (saline) > F-VCR > VCR-P NPs > VCR-Fol NPs > VCR-Fol/R7 NPs. When the treatment ended, the isolated tumor weights of different VCR formulation treated groups were all markedly smaller than that of the control (saline) group, and followed the same order (Figures 7A, 7C). The specific tumor inhibition rates of F-VCR, VCR-P NP, VCR-Fol NP and VCR-Fol/R7 NP groups were 32.58%, 37.08%, 51.69% and 79.78%, respectively. It was found that there was no significant difference between VCR-P NP and F-VCR treated groups. Though the passive targeting effect of unmodified NPs partly increased accumulation at the tumor site, without pH-sensitivity of VCR-P NPs did not improve the therapeutic effect for the tumor, which was connected with the relatively slower release of VCR from VCR-P NPs. Obviously, the tumor volume and weight of the VCR-Fol NP treated group showed a significant difference compared with that of the VCR-P NP treated group (P < 0.05) and VCR-Fol/R7 NP

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CONCLUSIONS In this study, pH-sensitive PLGA-PEG−folate and PLGA-PEGR7 were successfully synthesized and used as functional 892

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polymers to fabricate Fol/R7 NPs. The multifunctional nanoparticles were systematically evaluated in vitro and in vivo, which could target to MCF-7/Adr tumor precisely through targeting effect, then further increase cellular uptake and overcome multidrug resistance via folate receptor-mediated endocytosis and strong intracellular penetration of R7, eventually improving antitumor effect by acidic microenvironment-triggered drug rapid release. These results indicated that Fol/R7 NPs would be a promising nanocarrier for an antitumor drug such as vincristine sulfate (VCR) to overcome multidrug resistance and improve therapeutic efficacy.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-34204049. Fax: +86-21-34204049. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Basic Research Program of China (973 Program, Grant No. 2007CB936004) and the National Natural Science Foundation of China (Grant No. 30973644).

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